70 results found
Kaci FN, Lepore A, Papa S, et al., 2023, Screening Kinase-Dependent Phosphorylation of Key Metabolic Reprogramming Regulators., Methods Mol Biol, Vol: 2675, Pages: 205-218
Aerobic glycolysis has been commonly linked to cell proliferation, especially in cancer cells where it serves to generate sufficient energy and biosynthesis of new cell constituents needed for cell growth and division. The M2 isoform of pyruvate kinase (PKM2) catalyzes the last reaction of the glycolytic process. PKM2 promotes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, generating ATP and releasing pyruvate. This rate-limiting reaction relies therefore on the enzymatic activity of PKM2. The switching between the high- and low-activity states of PKM2 is subjected to a combination of allosteric mechanisms and fine-tuned regulation by oncogenes and tumor suppressor genes. These regulatory mechanisms involve primarily post-translational modifications of PKM2. Recent findings suggest that phosphorylation contributes to the regulation of PKM2 activity.Here, we describe an in vitro kinase assay we used to assess PKM2 phosphorylation by c-Jun N-terminal kinase (JNK), a master regulator of apoptosis, cell proliferation, and differentiation. While the use of phospho-specific antibodies gives information in terms of measuring the effects of a given kinase on its substrate, specific antibodies for newly identified phospho-groups are not readily available. The in vitro kinase assay allows the immediate measuring of phosphorylation of any substrate of interest. Although there are several options that do not use radioactive materials, we continue to rely on this biochemical method for robust quantitation of results. More interestingly, this protocol can be easily adapted to measure the activity of other kinases by using their specific substrates.
Lepore A, Kaci FN, Bubici C, et al., 2023, An Integrated Methodology to Quantify the Glycolytic Stress in Plasma Cell Myeloma in Response to Cytotoxic Drugs., Methods Mol Biol, Vol: 2675, Pages: 285-296
Multiple myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). Myeloma plasma cells, like many other cancer cells, change their metabolism in response to internal and external stimuli. The main metabolic alterations of MM cells include deregulated glycolysis (commonly associated with enhanced uptake and utilization of glucose), lipid metabolism dysregulation, as well as deregulated mitochondrial respiration (commonly associated with the deregulated formation of reactive oxygen species). Over the past decade, the discovery of novel methodologies and the commercialization of sophisticated instrumentation and reagents have facilitated the detection of real-time changes in cellular bioenergetics. Of those, the Seahorse™ extracellular flux (XF) analyzer has been widely used to evaluate the glycolytic flux and mitochondrial respiration in many cell types. While adherent cell lines are easy to use with this technology, non-adherent suspension cells are more difficult to handle especially when their metabolic activities are being investigated in response to drug treatment. Here, we provide an integrated protocol that allows the detection of extracellular acidification rate (ECAR) of live myeloma plasma cells in response to chemotherapeutic drugs. Our optimized protocol consists of treating myeloma cells with cytotoxic drug of interest in a standard culture plate prior to the real-time analysis in the XF analyzer. Furthermore, we provide results of experiments in which the metabolic activities of myeloma cells in response to cytotoxic treatment were compared between the manufacturer's basic procedure and our optimized protocol. Our observations suggest that our integrated protocol can be used to achieve consistent, well-standardized results and thus it may have broad applications in studies focusing on the characterization of metabolic events in non-adherent suspension cells.
Lepore A, Choy PM, Lee NC, et al., 2021, Phosphorylation and stabilization of PIN1 by JNK promote intrahepatic cholangiocarcinoma growth, Hepatology, Vol: 74, Pages: 2561-2579, ISSN: 0270-9139
BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and thus the key downstream effectors of this pathway remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signalling in ICC that could open new therapeutic opportunities. APPROACH AND RESULTS: Using loss- and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumours, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 via all-trans retinoic acid (ATRA), an FDA-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation via PIN1 inhibition.
Papa S, Bubici C, Syn W-K, 2021, STARD1: a new rising StAR in cholesterol-mediated hepatocarcinogenesis, Hepatobiliary Surgery and Nutrition, Vol: 10, ISSN: 2304-3881
Arif E, Wang C, Swiderska-Syn MK, et al., 2021, Targeting myosin 1c inhibits murine hepatic fibrogenesis, American Journal of Physiology: Gastrointestinal and Liver Physiology, Vol: 320, Pages: G1044-G105, ISSN: 0193-1857
Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, TGF-β stimulation to hepatic stellate cells (HSCs, the liver pericyte and key cell type responsible for the deposition of extracellular matrix upregulates Myo1c expression, while genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β induced fibrogenic responses, resulting in repression of α-SMA and Col1α1 mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response and contractility when compared with vehicle treated HSCs. Importantly, phosphorylation of SMAD2 and SMAD3 were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c-KO MEFs upon TGF-β stimulation. Using the genetic Myo1c knockout (Myo1c-KO) mice, we confirmed that Myo1c is critical for fibrogenesis as Myo1c-KO mice were resistant to CCl4 induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c-mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.
Flati V, Corsetti G, Papa S, 2021, Editorial: the dynamic interplay between nutrition, autophagy and cell metabolism, Frontiers in Cell and Developmental Biology, Vol: 9, Pages: 1-3, ISSN: 2296-634X
Bubici C, Lepore A, Papa S, 2019, ASKing no more: the emerging role of DUSP12 in the regulation of hepatic lipid metabolism, Hepatology, Vol: 70, Pages: 1091-1094, ISSN: 0270-9139
Accumulation of fat in liver cells not due to alcohol abuse is the hallmark of non-alcoholic fatty liver disease (NAFLD), a common condition that may progress to non-alcoholic steatohepatitis (NASH) characterized by liver inflammation.(1) Over a long period of time, NASH may lead to fibrosis with consequent cirrhosis, which in turn predisposes patients to hepatocellular carcinoma.
Bubici C, Papa S, 2019, Editorial: The warburg effect regulation under siege: the intertwined pathways in health and disease, Frontiers in Cell and Developmental Biology, Vol: 7, ISSN: 2296-634X
Papa S, Choy PM, Bubici C, 2019, The ERK and JNK pathways in the regulation of metabolic reprogramming, Oncogene, Vol: 38, Pages: 2223-2240, ISSN: 0950-9232
Most tumor cells reprogram their glucose metabolism as a result of mutations in oncogenes and tumor suppressors, leading to the constitutive activation of signaling pathways involved in cell growth. This metabolic reprogramming, known as aerobic glycolysis or the Warburg effect, allows tumor cells to sustain their fast proliferation and evade apoptosis. Interfering with oncogenic signaling pathways that regulate the Warburg effect in cancer cells has therefore become an attractive anticancer strategy. However, evidence for the occurrence of the Warburg effect in physiological processes has also been documented. As such, close consideration of which signaling pathways are beneficial targets and the effect of their inhibition on physiological processes are essential. The MAPK/ERK and MAPK/JNK pathways, crucial for normal cellular responses to extracellular stimuli, have recently emerged as key regulators of the Warburg effect during tumorigenesis and normal cellular functions. In this review, we summarize our current understanding of the roles of the ERK and JNK pathways in controlling the Warburg effect in cancer and discuss their implication in controlling this metabolic reprogramming in physiological processes and opportunities for targeting their downstream effectors for therapeutic purposes.
Papa S, Lee NC, Bubici C, 2018, Deciphering preventive and prognostic biomarkers of liver cancer, NCRI Cancer Conference 2018, Publisher: National Cancer Research Institute
Lee NCW, Carella MA, Papa S, et al., 2018, High expression of glycolytic genes in cirrhosis correlates with the risk of developing liver cancer, Frontiers in Cell and Developmental Biology, Vol: 6, ISSN: 2296-634X
A marked increase in the rate of glycolysis is a key event in the pathogenesis of hepatocellular carcinoma (HCC), the main type of primary liver cancer. Liver cirrhosis is considered to be a key player in HCC pathogenesis as it precedes HCC in up to 90% of patients. Intriguingly, the biochemical events that underlie the progression of cirrhosis to HCC are not well understood. In this study, we examined the expression profile of metabolic gene transcripts in liver samples from patients with HCC and patients with cirrhosis. We found that gene expression of glycolytic enzymes is up-regulated in precancerous cirrhotic livers and significantly associated with an elevated risk for developing HCC. Surprisingly, expression levels of genes involved in mitochondrial oxidative metabolism are markedly increased in HCC compared to normal livers but remain unchanged in cirrhosis. Our findings suggest that key glycolytic enzymes such as hexokinase 2 (HK2), aldolase A (ALDOA), and pyruvate kinase M2 (PKM2) may represent potential markers and molecular targets for early detection and chemoprevention of HCC.
Papa S, Bubici C, 2018, Feeding the hedgehog: a new meaning for JNK signalling in liver regeneration, Journal of Hepatology, Vol: 69, Pages: 572-574, ISSN: 0168-8278
Manka P, Coombes JD, Boosman R, et al., 2018, Thyroid hormone in the regulation of hepatocellular carcinoma and its microenvironment, Cancer Letters, Vol: 419, Pages: 175-186, ISSN: 0304-3835
Hepatocellular carcinoma (HCC) commonly arises from a liver damaged by extensive inflammation and fibrosis. Various factors including cytokines, morphogens, and growth factors are involved in the crosstalk between HCC cells and the stromal microenvironment. Increasing our understanding of how stromal components interact with HCC and the signaling pathways involved could help identify new therapeutic and/or chemopreventive targets. It has become increasingly clear that the cross-talk between tumor cells and host stroma plays a key role in modulating tumor growth. Emerging reports suggest a relationship between HCC and thyroid hormone signaling (dysfunction), raising the possibility that perturbed thyroid hormone (TH) regulation influences the cancer microenvironment and cancer phenotype. This review provides an overview of the role of thyroid hormone and its related pathways in HCC and, specifically, its role in regulating the tumor microenvironment.
Verzella D, Bennett J, Fischietti M, et al., 2018, GADD45β loss ablates innate immunosuppression in cancer, Cancer Research, Vol: 78, Pages: 1275-1292, ISSN: 1538-7445
T cell exclusion from the tumour microenvironment (TME) is a major barrier to overcoming immune escape. Here we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45β that restricts tumour-associated inflammation and T cell trafficking into tumours. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma (HCC) and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of pro-inflammatory tumour-associated macrophages (TAM) and intratumoural immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Further, they suggest a therapeutic target in GADD45β for re-programming TAM to overcome immunosuppression and T cell exclusion from the TME.
Briones MA, Coombes JD, Mellone M, et al., 2016, The role of osteopontin isoforms in cholangiocarcinoma, 67th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD), Publisher: WILEY, Pages: 235A-235A, ISSN: 0270-9139
, 2016, Poster Session I (Abstracts 259 – 729), Hepatology, Vol: 64, Pages: 136-361, ISSN: 0270-9139
Papa S, Bubici C, 2016, Linking apoptosis to cancer metabolism: Another missing piece of JuNK, Molecular & Cellular Oncology, Vol: 3, ISSN: 2372-3556
Cancer cells become dependent on aerobic glycolysis to sustain rapid proliferation and escape apoptosis. How this metabolic change, also known as the Warburg effect, is linked to apoptosis remains largely unknown. Our new data place c-Jun N-terminal kinase in the center of a hub regulating apoptosis and cancer metabolism.
Briones-Orta MA, Coombes JD, Mellone M, et al., 2015, Osteopontin-c isoform promotes a mesenchymal phenotype in human cholangiocarcinoma cells, 66th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD), Publisher: WILEY-BLACKWELL, Pages: 1171A-1171A, ISSN: 0270-9139
, 2015, Poster Session 4: Experimental Hepatocarcinogenesis; Hepatitis B: Treatment, Hepatology, Vol: 62, Pages: 1151A-1181A, ISSN: 0270-9139
Papa S, Bubici C, 2015, Starving cancer cells of sugar could be the key to future treatment
Iansante V, Choy PM, Fung SW, et al., 2015, PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation, Nature Communications, Vol: 6, ISSN: 2041-1723
Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.
Iansante V, Choy PM, Chokshi S, et al., 2015, Addressing the interplay between apoptosis and glucose metabolism in liver cirrhosis and HCC, Digestives Disorders Federation
Choy PM, Sufi J, Glaser S, et al., 2015, INHIBITION OF MAPK SIGNALLING PROMOTES CELL CYCLE ARREST AND SENSITISES INTRAHEPATIC CHOLANGIOCARCINOMA CELLS TO CHEMOTHERAPY, 2nd Digestive-Disorders-Federation Conference, Publisher: BMJ PUBLISHING GROUP, Pages: A458-A458, ISSN: 0017-5749
Iansante V, Choy PM, Chokshi S, et al., 2015, INCREASED AEROBIC GLYCOLYSIS IS ASSOCIATED WITH POOR OUTCOME AND SUPPRESSION OF APOPTOSIS IN HUMAN LIVER CIRRHOSIS AND HCC, 50th International Liver Congress of the European-Association-for-the-Study-of-the-Liver, Publisher: ELSEVIER SCIENCE BV, Pages: S427-S427, ISSN: 0168-8278
Coombes JD, Swiderska-Syn M, Dollé L, et al., 2015, Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice, Gut, Vol: 64, Pages: 1120-1131
Iansante V, Choy PM, Fung SW, et al., 2014, UPREGULATION OF A NOVEL PROTEIN IN HCC ENHANCES CANCER CELL SURVIVAL BY SUPPRESSING SPECIFIC APOPTOTIC EFFECTORS, 49th Annual International Liver Congress of the European-Association-for-the-Study-of-the-Liver, Publisher: ELSEVIER SCIENCE BV, Pages: S89-S89, ISSN: 0168-8278
Briones-Orta MA, Coombes JD, Kitamura N, et al., 2014, Osteopontin isoforms are upregulated in human cholangiocarcinoma cells and modulate levels of the TGF-beta repressor, SnoN, 65th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases, Publisher: WILEY-BLACKWELL, Pages: 818A-818A, ISSN: 0270-9139
Bubici C, Papa S, 2014, JNK signalling in cancer: in need of new, smarter therapeutic targets, BRITISH JOURNAL OF PHARMACOLOGY, Vol: 171, Pages: 24-37, ISSN: 0007-1188
Wang Z, Hou J, Lu L, et al., 2013, Small Ribosomal Protein Subunit S7 Suppresses Ovarian Tumorigenesis through Regulation of the PI3K/AKT and MAPK Pathways, PLOS ONE, Vol: 8, ISSN: 1932-6203
Coombes J, Claridge LC, Swiderska-Syn M, et al., 2013, Osteopontin enhances liver progenitor cell responses in progressive Nonalcoholic Steatohepatitis, 64th Annual Meeting and Postgraduate Course of the American-Association-for-the-Study-of-Liver-Diseases, Publisher: WILEY-BLACKWELL, Pages: 535A-535A, ISSN: 0270-9139
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